1. Field of the Invention
The invention in the fields of microbiology and medicine relates to methods for rapid early detection of mycobacterial disease in humans based on the presence of antibodies to particular xe2x80x9cearlyxe2x80x9d mycobacterial antigens which have not been previously recognized for this purpose. Assay of such antibodies on select partially purified or purified mycobacterial preparations containing such early antigens permits diagnosis of TB earlier than has been heretofore possible. Also provided is a surrogate marker for screening populations at risk for TB, in particular subjects infected with human immunodeficiency virus (HIV).
2. Description of the Background Art
Recent estimates by the World Health Organization (WHO) suggest that approximately 90 million new cases of tuberculosis (xe2x80x9cTBxe2x80x9d) will occur during this decade leading to about 30 million deaths (Raviglione, M. C. et al., 1995, JAMA. 273:220-226). The spread of HIV in populations already having a high incidence of TB related to socioeconomic factors and malnutrition has resulted in a resurgence of TB all over the world (Raviglione, M. C. et al., 1992, Bull World Health Organ 70:515-526; Harries A. D., 1990, Lancet. 335:387-390). This resurgence has renewed interest in developing improved vaccines, diagnostics, drugs and drug delivery regimens for TB. Furthermore, the immune dysfunction caused by HIV infection leads to a high rate of reactivation of latent TB, increased susceptibility to primary disease, as well as an accelerated course of disease progression (Raviglione et al., 1992, supra; 1995, supra; Shafer R. W. et al., 1996, Clin. Infect. Dis. 22:683-704; Barnes P. F. et al., 1991, N. Engl. J. Med. 324:1644-1650; Selwyn P. A. et al., 1989, N. Engl. J. Med. 320:545-550).
It is well established that cellular immunity is critical for protection against TB. Much of the work in this field is focused on defining the antigens of the causative bacterium, Mycobacterium tuberculosis (M. tuberculosis; also abbreviated herein as xe2x80x9cMtxe2x80x9d) that can elicit effective immunity and on understanding the role of various cell populations in host-pathogen interactions (Andersen, P. et al., 1992, Scand. J. Immunol. 36:823-831; Havlir, D. V. et al., 1991, Infect. Immun. 59:665-670; Orme, I. M. et al., 1993, J. Infect. Dis. 167:1481-1497).
Delayed hypersensitivity measured as cutaneous immune reactivity to a purified protein derivative of Mt (abbreviated xe2x80x9cPPDxe2x80x9d) is the only marker available for detection of latent infection with Mt. However, the sensitivity of the PPD skin test is substantially reduced during HIV infection (Raviglione et al., 1992, supra, 1995, supra; Graham N. M. H. et al., 1991, JAMA 267:369-373; Huebner R. E. et al., 1994, Clin. Infect. Dis. 19:26-32; Huebner R. E. et al., 1992, JAMA 267:409-410; Caiaffa W. T. et al., 1995, Arch. Intern. Med. 155:2111-2117). Furthermore, vaccination with a closely related mycobacterium designated Bacillus Cahnette-Guerin (BCG) or previous exposure to other mycobacterial species can lead to false positive results in a PPD skin test. Not only does PPD reactivity fail to distinguish active, subclinical disease from latent infection, but the time between a positive skin test and development of clinical disease may range from months to several years (Selwyn P. A. et al., supra).
Because of the susceptibility of immunocompromised individuals to TB, the U.S. Centers for Disease Control and Prevention recommends preventive isoniazid therapy for all HIV seropositive (HIV+), PPD-positive (PPD+) individuals. However, the optimal time for such therapy is not clear and, ideally, should coincide with replication of previously latent bacteria. Unnecessary therapy must be minimized because prolonged isoniazid treatment can have serious toxic side effects (Shafer et al., supra). The impact of such treatment on emergence of drug resistant bacteria is still unclear. The use of preventive therapy in developing countries is seriously limited by the high frequency of PPD+ individuals coupled with the lack of adequate medico-social infrastructure and economic resources. High risk populations are also found in the United States, primarily intravenous drug users, homeless people, prison inmates and residents of slum areas (Fitzgerald, J. M. et al., 1991, Chest 100:191-200; Graham, N. M. H. et al., 1992, JAMA 267:369-373; Friedman, L. N. et al., 1996, New Engl. J. Med. 334:828-833) as well as household contacts of TB patients. Thus, discovery of additional surrogate markers for early detection and prompt treatment of active, subclinical TB in such high risk populations is urgently required.
Antibody responses in TB have been studied for several decades primarily for the purpose of developing serodiagnostic assays. Although some seroreactive antigens/epitopes have been identified, interest in antibody responses to M. tuberculosis has waned because of the lack of progress in simple detection of corresponding antibodies. Studies using crude antigen preparations revealed that healthy individuals possess antibodies that cross-react with several mycobacterial antigens. Such antibodies are believed to have been elicited by exposure to commensal and environmental bacteria and vaccinations (Bardana, E. J. et al., 1973, Clin. Exp. Immunol. 13:65-77; Das, S. et al., 1992, Clin. Exp. Immunol. 89:402-406; Del Giudice, G. et al., 1993, J. Immunol. 150:2025-2032; Grange, J. M., 1984, Adv. Tuberc. Res. 21:1-78; Havlir, D. V. et al., supra; Ivanyi, J. et al., 1989, Brit. Med. Bull. 44:635-649; Verbon, A. et al., 1990, J. Gen. Microbiol. 136:955-964). During the last decade, several mycobacterial antigens have been isolated and characterized (Young, D. B. et al., 1992, Mol. Microbiol. 6:133-145), including the 71 kDa DnaK, 65 kDa GroEL, 47 kDa elongation factor tu, 44 kDa PstA homologue, 40 kDa L-alanine dehydrogenase, 38 kDa PhoS, 23 kDa superoxide dismutase, 23 kDa outer membrane protein, 12 kDa thioredoxin, and the 14 kDa GroES. However, a majority of the antigens identified so far bear significant homology to the analogous proteins in other mycobacteria and non-mycobacterial prokaryotes (Andersen, A. B. et al., 1992, Infect. Immun. 60:2317-2323; Andersen, A. B. et al., 1989, Infect. Immun. 57:2481-2488; Braibant, M. et al., 1994, Infect. Immun. 62:849-854; Carlin, N. I. A. et al., 1992, Infect. Immun. 60:3136-3142; Garsia, R. J. et al., 1989, Infect. Immun. 57:204-212; Hirschfield, G. R. et al., 1990, J. Bacteriol. 172:1005-1013; Shinnick, T. M. et al., 1989, Nucl. Acids Res. 1 7:1254; Shinnick, T. M. et al., 1988, Infect. Immun. 56:446-451; Wieles, B. et al., 1995, Infect. Immun. 63:4946-4948; Young, D. B. et al., supra; Zhang, Y. et al., 1991, Mol. Microbiol. 5:381-391). Thus, almost all individuals (healthy or diseased) have antibodies to epitopes of conserved regions of these antigens. These antibodies are responsible for the uninformative (and possibly misleading) cross-reactivity observed with crude Mt antigen preparations (Davenport, M. P. et al., 1992, Infect. Immun. 60:1170-1177; Grandia, A. A. et al., 1991, Immunobiol. 182:127-134; Meeker, H. C. et al., 1989, Infect. Immun. 57:3689-3694; Thole, J. et al., 1987, Infect. Immun. 55:1466-1475).
Because such cross-reactive antibodies would mask the presence of antibodies specific for Mt antigens, some of the purified antigens such as the 38 kDa PhoS, the 30/31 kDa xe2x80x9cantigen 85xe2x80x9d (discussed in more detail below), 19 kDa lipoprotein, 14 kDa GroES and lipoarabinomannan have been prepared and tested (Daniel, T. et al., 1985 Chest. 88:388-392; Drowart, L. et al., 1991, Chest. 100:685-687; Jackett, P. S. et al., 1988, J. Clin. Microbiol. 26:2313-2318; Ma, Y. et al., 1986, Am. Rev. Respir. Dis. 134:1273-1275; Sada, E. et al., 1990, J. Clin. Microbiol. 28:2587-2590; Sada, E. D. et al., 1990, J. Infect. Dis. 162:928-931; Van Vooren, J. P. et al., 1991, J. Clin. Microbiol. 29:2348-2350). It is noteworthy that the choice of which antigen to test was dictated primarily by (a) its availability, (b) its immunodominance in animal immunizations, or (c) ease of its biochemical purification. None of these criteria take into account the reactivity of the antigen which occurs naturally in the human immune response to mycobacterial diseases. Use of the 38 kDa antigen has provided the highest serological sensitivity and specificity so far (Daniel, T. M. et al., 1987, Am. Rev. Respir. Dis. 135:1137-1151; Harboe, M. et al., 1992, J. Infect. Dis. 166:874-884; Ivanyi, J. et al., 1989, supra). However, in contrast to the present invention, the presence of anti-38 kDa antibodies is associated primarily with treated, advanced and recurrent TB (Bothamley, G. H. et al., 1992, Thorax. 47:270-275; Daniel, T. M. et al., 1986, Am. Rev. Respir. Dis. 134:662-665; Ma, Y. et al., 1986, Am. Rev. Respir. Dis. 134:1273-1275).
One convention in mycobacterial protein nomenclature is the use of MPB and MPT numbers. MPB denotes a protein purified from M. bovis BCG followed by a number denoting its relative mobility in 7.7% polyacrylamide gels at a pH of 9.5. MPT denotes a protein isolated from M. tuberculosis. In proteins examined prior to this invention, no differences in the N-terminal amino acid sequence were shown between these two mycobacterial species.
Wiker and colleagues have studied a family of secreted Mt proteins which include a complex of 3 proteins termed antigens 85A, 85B and 85C (also known as the xe2x80x9c85 complexxe2x80x9d or xe2x80x9c85cxxe2x80x9d) (Wiker, H. G. et al., 1992, Scand. J. Immunol. 36:307-319; Wiker, H. G. et al., 1992, Microbiol. Rev. 56:648-661). This complex was originally found in M. bovis BCG preparations which produced a secreted antigen comprising a complex of three closely related components, antigen 85A, 85B, and 85C (Wiker, H. G. et al. 1986, Int. Arch. Allergy Appl. Immunol. 81:289-306). The corresponding components of Mt are also actively secreted. The 85 complex is considered the major secreted protein constituent of mycobacterial culture fluids though it is also found in association with the bacterial surface. In most SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, 85A and 85C are not properly resolved, whereas isoelectric focusing resolves three distinct bands.
Genes encoding six of the secreted proteins: 85A, 85B, 85C, xe2x80x9cantigen 78xe2x80x9d (usually referred to as the 38 kDa protein), MPB64 and MPB70 have been cloned. Three separate genes located at separate sites in the mycobacterial genome encode 85A, B and C (Content, J. et al., 1991, Infect. Immun. 59:3205-3212). A gene encoding the antigen known as MPT-32 (reported as a 45/47 kDa secreted antigen complex) has been cloned, sequenced and expressed (Laqueyrerie, A. et al. , 1995, Infec. Immun. 63:4003-4010) and designated as the apa gene. One of the present co-inventors and his collaborators provided evidence for glycosylation sites on this protein (Dobos, K. M. et al., 1996, J. Bacteriol. 178:2498-2506 and Example III herein). However, the need continues for further elucidation of the biochemistry and immunochemistry of Mt proteins and glycoproteins which are potentially important as serodiagnostic tools. The fall definition of glycosylation sites and the nature and extent of glycosylation of glycosylated proteins has been scant. Initial evidence for the presence of glycoproteins in Mt was based on the observation of discrete concanavalin A (ConA)-binding products upon PAGE and electroblotting of protein preparations. However, since these patterns occurred in the midst of considerable quantities of mannose (Man)-containing lipoglycans and phospholipids (Dobos et al., supra), chemical proof of amino acid glycosylation is still considered necessary and is provided as part of this invention.
The antigen 85 complex is often referred to as the xe2x80x9c30/31 kDa doublet,xe2x80x9d although slightly different molecular mass designations have been reported. The following list shows the molecular masses of the individual components of antigen 85 complex plus two additional antigens (in SDS-PAGE) as described by Wiker and colleagues, along with alternative nomenclatures:   "AutoLeftMatch"                              Ag85A          =                      MPT44            =                          xe2x80x83                        ⁢                          31              ⁢                              xe2x80x83                            ⁢              kDa                                                                    Ag85B          =                      MPT59            =                          xe2x80x83                        ⁢                          30              ⁢                              xe2x80x83                            ⁢              kDa                                                                    Ag85C          =                      MPT45            =                          xe2x80x83                        ⁢                          31.5              ⁢                              xe2x80x83                            ⁢              kDa                                                                    MPT64          =                      xe2x80x83                    ⁢                      26            ⁢                          xe2x80x83                        ⁢            kDa                                                        MPT51          =                      xe2x80x83                    ⁢                      27            ⁢                          xe2x80x83                        ⁢            kDa                                                                    Ag78            ⁢                          xe2x80x83                        ⁢            ⋯                    ⁢                      xe2x80x83                    =                      xe2x80x83                    ⁢                      38            ⁢                          xe2x80x83                        ⁢            kDa                                                        MPT32          =                      xe2x80x83                    ⁢                                    45              /              47                        ⁢                          xe2x80x83                        ⁢            kDa            ⁢                          xe2x80x83                        ⁢                          (found to be 38 / 42   kDa
 by the present inventors)                                          
Wiker""s group studied cross-reactions between five actively secreted Mt proteins by crossed immunoelectrophoresis, SDS-PAGE with immunoblotting and enzyme immunoassay (EIA) using (1) polyclonal rabbit antisera to the purified proteins and (2) a mouse monoclonal antibody (xe2x80x9cmAbxe2x80x9d). The mAb HBT4 reacted with the MPT51 protein. The 85A, 85B, and 85C constituents cross-reacted extensively, though each had component-specific in addition to cross-reacting epitopes. These components also cross-reacted with MPT51 and MPT64. Amino acid sequence homology was shown between 85A, 85B, 85C and MPT51. MPT64 showed less homology. Striking homology was also found between two different structures within the 85B sequence. Thus a family of at least four secreted proteins with common structural features has been demonstrated in mycobacteria. Three of these proteins bind readily to fibronectin (Abou-Zeid, C., 1988, Infect. Immun. 56:3046-3051; Abou-Zeid, C., 1988, Infect. Immun. 59:2712-2718; Harboe, M. et al., 1992, Clin. Inf. Dis. 14:313-319).
The aligned amino acid sequences listed below illustrate the homology of a fragment of 85A, 85B, 85C, MPT51 and MPT64. The numbers at the top correspond to the part of the sequence shown. The N-terminal sequences were determined on isolated proteins and aligned by visual inspection. The sequence from position 66 to 91 of MPT64 is the sequence deduced from the cloned gene.
The N-terminal sequence of MPT51 showed 72% homology with the sequence of the Ag 85 components (when P at position 2 is aligned with P at position 7 of the three Ag 85 components.
Apart from fibronectin binding, little information concerning the primary functions of antigen 85 complex proteins is available. Although the art has not considered antibodies as playing a significant role in protective immunity against mycobacterial infections, Wiker et al. (supra) speculated that the existence of interactions between Ag 85 and fibronectin implied that an antibody to Ag 85 which could block this interaction might affect early events in disease progression and increase host resistance.
Studies of TB patients showed that assays of antibodies to the Ag 85 complex had a sensitivity of about 50%. With regard to specificity, the Ag 85 components are highly cross-reactive so that positive responses are expected (and found) in healthy controls, particularly in geographic areas of high exposure to atypical mycobacteria. The different degree of specificity is thus highly dependent on the kind of control subjects used. It is noteworthy that traditional BCG vaccination does not appear to induce a significant antibody response, though it is interesting that antibodies to mycobacterial antigens increased when anti-TB chemotherapy was initiated.
C. Espitia et al., 1989, Clin Exp Immunol 77:373-377, found antibodies to the 30/31 kDa doublet band (presumably 85A and 85C) in 55.9% of TB patient sera (and in 56.5% of lepromatous leprosy sera). Sera from healthy individuals often showed binding which was weaker than TB patients. Van Vooren, J. P. et al., 1991, J. Clin. Microbiol. 29:2348-2350, found that antigen 85A reacted with sera from tuberculous as well as nontuberculous individuals. By contrast, 85B and 85C did not react with the control sera but reacted with 20 of 28 serum samples (71%) from tuberculous patients. Wiker and colleagues concluded that the future of the serology of antibody responses to antigen 85 would require investigation of antibodies to component-specific epitopes and in particular to species-specific epitopes. The extensive cross-reactivity of antigen 85 in different species of mycobacteria suggested to Wiker et al. (supra) that tests could attain sufficient sensitivity, though suitable mAbs were said to be essential for further development of tests for infection with Mt (and atypical mycobacteria). Importantly, the present inventors note the deficiency in the art of analysis of antibodies at different stages of disease. This is one of the primary deficiencies addressed by this invention.
C. Espitia et al., 1995, Infect. Immun. 63:580-584, found reciprocal cross-reactivity between a Mt 50/55 kDa protein and a M. bovis BCG 45/47 kDa antigen using a rabbit polyclonal antiserum against the M. bovis protein and a mAb against the Mt antigen. Both antigens were secreted glycoproteins. The N-terminal sequences and total amino acid content of these proteins were very similar. Analysis by 2D gel electrophoresis showed at least seven different components in the Mt 50/55 kDa antigen. In solid-phase immunoassays, purified Mt 50/55 kDa protein was recognized by sera from 70% of individuals (n=77) with pulmonary TB. The N-terminus of the Mt 41 kDa antigen known as MPT32 was very similar to the N-termini of the 50/55 kDaxe2x80x94and the 45-47 kDa proteins. The molecular mass of this Mt protein was deduced to be 45-47 kDa. Espitia et al., supra, speculated about a diagnostic potential for these antigens based on their observation of antibodies in 70% of their TB patients. However, the potential of this antigen as an early diagnostic agent for TB was neither analyzed nor even suggested.
In sum, none of the antigens studied so far, with the possible exception of MPT32 (as will be described herein) has emerged as a suitable candidate for development of a diagnostic assay for early stages of TB. Since antigens/epitopes recognized during natural infection and disease progression in humans may differ substantially from those recognized by animals upon artificial immunization (Bothamley, G. et al., 1988, Eur. J. Clin. Microbiol. Infect. Dis. 7:639-645; Calle, J. et al., 1992, J. Immunol. 149:2695-2701; Hartskeerl, R. A. et al., 1990, Infect. Immun. 58:2821-2827; Laal, S. et al., 1991, Proc. Natl. Acad. Sci. USA. 88:1054-1058; Meeker, H. C. et al., 1989, Infect. Immun. 57:3689-3694; Verbon, A., 1994, Trop. Geog. Med. 46:275-279), there is a pressing need in the art for selection of antigens based on their ability to stimulate the human immune system. This would permit the identification of such useful antigens and design of diagnostic assays for early detection of TB.
TB in HIV Infected Subjects
Studies aimed at determining the integrity of humoral immune memory during HIV infection have shown that the ability to respond to recall antigens by producing significant amounts of high-affinity specific IgG antibodies was maintained during the time prior to onset of clinical AIDS (Janoff, E. N. et al., 1991, J. Immunol 147:2130-2135). Secondary antibody responses are relatively independent of T cell help, and B cells specific for recall antigens are present in normal frequency in HIV-infected individuals (Janoff et al. (supra); Kroon F. P. et al., 1995, Clin. Infect. Dis. 21:1197-1203). Comparison of secondary responses to different antigens in HIV-infected individuals also suggested that the level of immunologic memory established prior to HIV infection may influence the ability of the subject to respond post-infection (Janoff et al. (supra)). Since TB in HIV-infected individuals often results from reactivation of latent infection, and reactivated TB is known to occur relatively early during the course of HIV disease progression, the immune system may be sufficiently intact to generate antibody responses towards bacteria emerging from latency. If this occurs, HIV-infected subjects with active TB infection should have detectable antibodies directed towards Mt antigens.
Although the literature on TB infection in subjects not infected with HIV is extensive, reports on antibody responses of HIV/TB patients to M. tuberculosis, have been scant and controversial. Farber, C. et al., 1990, J. Infect. Dis, 162:279-280, reported the presence of antibodies to the p32 antigen (same as 85A) in 7 of 8 HIV/TB patients. Da Costa, C. et al., 1993, Clin. Exp. Immunol. 91:25-29, reported the presence of anti-lipoarabinomannan (LAM) antibodies in 35% of such patients. Barer, L. et al., 1992, Tuber. Lung. Dis. 73:187-191, reported anti-PPD antibodies in 36% of HIV/TB patients. Martin-Casabona, N. et al., 1992, J. Clin. Microbiol. 30:1089-1093, reported anti-sulfolipid (SLIV) antibodies in 73% of their patients. In addition, van Vooren, P. et al., 1988, Tubercle. 69:303-305, reported that anti-p32 antibodies were detectable in an HIV/TB patient for several months prior to clinical manifestation of TB. In contrast, analysis of responses to Ag60 (Saltini C. et al., 1993, Am. Rev. Respir. Dis. 145:1409-1414; van der Werf, T. S. et al., 1992. Med Microbiol Immunol 181:71-76) and Ag85B (McDonough, J. A. et al., 1992, J. Lab. Clin. Med. 120:318-322) failed to detect antibodies in these patients.
Hence, there is a particular need in the art for methods to detect TB infections at early stages in HIV patients since they comprise one of the largest populations at risk for TB throughout the world.
A number of laboratories have reported on antibodies, mainly to infectious agents, in urine. For example, Takahashi S; et al. (Clin Diagn Lab Immunol, 1998, 5:24-27) found antibodies to rubella virus in urine and serum samples from healthy individuals who underwent rubella vaccination. Shutov AM et al. Arkh (RUSSIA) 1996, 68:35-37 detected antibodies in urine to the virus causing hemorrhagic fever with renal syndrome (HFRS) and concluded that detection of antibodies to the virus both in the blood and urine can be used for earlier diagnosis Vereta LA; et al. (Vopr Virusol (RUSSIA) 1993, 38:18-21) used a a commercial diagnostic indirect imunofluorescence assay to detection antibodies to the hanta virus in the urine of patients with HFRS.
Koopmans M et al. (J Med Virol, 1995, 46:321-328) demonstrated presence of antibodies to human cytomegalovirus (HCMV) in urine samples by ELISA and immunoblot using a recombinant form of the major antigenic protein of HCMV designated pp150). The presence of HCMV antibodies correlated significantly with congenital infection (as detected by tissue culture isolation of virus from urine samples of newborns), especially with asymptomatic cases (sensitivity 70%; specificity 94%). The authors interpreted this reflect a local (renal) immune response to HCMV in congenitally infected children, which may have diagnostic applications.
Zhang X et al. (J Med Virol, 1994,44:187-191) used commercial immunoassays to detect antibodies to hepatitis C virus (HCV) in urine (229 serum/urine matched samples from forensic autopsy cases). The urine samples produced positive results on 44-45 of the 46 samples that were positive by conventional screening tests and by supplemental tests. Generally, five times the serum volume was required for the screening tests to be optimal for urine samples. The same group (Constantine N T et al., Am J Clin Pathol, 1994, 101:157-161) detected antibodies to HIV in urine.
A group of Japanese investigatos (Hashida S et al., J Clin Lab Anal, 1994, 8:237-246; Hashinaka K et al., J. Clin Microbiol 1994, 32:819-22; Hashida S et al., J Clin Lab Anal 1994, 8:149-156 Hashida S et al., J Clin Lab Anal 1994, 8:86-95) diagnosed HIV-1 infection in asymptomatic carriers by detecting IgG antibody to HIV-1 in urine using an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) with recombinant proteins as antigen. They reported that sensitivity could be improved by a longer assay of bound enzyme activity by using concentrated urine samples and by the combined use of thee different recombinant HIV antigens.
Urnovitz HB et al., (Lancet Dec. 11, 1993, 342:1458-9), discovered that 7 individuals who were negative for HIV-1 antibody in a licensed serum EIA were positive in a urine EIA and western blot (WB).
Connell J A et al., J Med Virol 1993, 41:159-64, described a rapid, simple, and robust IgG-capture enzyme-linked immunosorbent assay (GACELISA) suitable for the detection of anti-HIV 1 and 2 antibodies in saliva and urine The assay was 100% sensitive on 126 urine specimens collected from anti-HIV-positive patients and 100% patient on 422 uriney specimens collected from anti-HIV-negative individuals. An earlier study from this laboratory (Connell J A et al., Lancet, 1990, 335:1366-1369) described anti-HIV antibodies in urine by GACELISA). Gershy-Damet G M et al. Trans R Soc Trop Med Hyg 1992, 86:670-671, used these assays successfully for urinary diagnosis of HIV-1 and HIV-2 in Africa, using to unprocessed saliva and urine specimens. They found the assay to be as accurate as conventional EIAs on serum tested under similar conditions.
Perry K R et al., J Med Virol 1992, 38:265-270, detected IgG and IgM antibodies to hepatitis A and hepatitis B core antigens in urine specimens.
As an example of urinary autoantibodies, Ben-Yehuda A et al. (J Autoimmun, 1995, 8:279-291) found that urine of patients with active SLE and of MRL-1/1 mice contained antibodies that bind extracellular matrix, primarily the 200 kDa light chain of laminin. The level of the anti-laminin antibodies correlated with disease activity.
Dr. A. Friedman-Kien and his colleagues have examined paired urine and serum samples in a search for antibodies to hepatitis B surface anitgen (HBs), hepatitis B core antigen (HBc), CMV and HIV in paired urine and serum samples from the same HIV-infected individuals (Cao Y et al., 1989, AIDS. Res. Hum. Retrovir. 5:311). In all individuals with anti-HIV antibodies in serum, anti-HIV antibodies were found in their urine; no such correlation was observed for HBs and CMV antibodies. The anti-HIV urine antibodies were of the IgG class, and gp160 and gp120 were the most consistently recognized proteins. Based on these observations, a urine based diagnostic assay for HIV-1 was been developed at by these investigators.
In view of the prevalence of TB in the HIV-infected individuals, especially in the developing countries, and the risks and costs involved in collection of blood/serum for serodiagnosis, the present inventors evaluated the urine of TB patients for presence of anti-mycobacterial antibodies. They reasoned that since M. tuberculosis infects the mucosal surfaces in the lung, it may induce antibodies in mucosal tissues resulting in the presence of antibodies in the urine. The positive results of these studies are presented below. The ability to use urine as the sample material will make the test extremely attractive to public health officials and to industry.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
The present inventors have systematically analyzed the reactivity of sera and urine from TB patients with antigens from Mt to delineate the major targets of human antibody responses which occur relatively early in the progression of the infection to disease. They observed that initial immunoadsorption of patient sera with E. coli antigens successfully reduced interference by cross-reactive antibodies, thus allowing a new approach to serological studies. The immunoadsorbed sera allowed identification of a number of antigens of Mt that are recognized by antibodies in a large proportion of patients, and during earlier stages of disease progression. These antigens are therefore useful tools in methods of diagnosing TB. Prominent among these antigens is a high molecular weight secreted protein of 88 kDa or 85 kDa (depending on conditions as will be described below). This protein is termed xe2x80x9cthe 88 kDa proteinxe2x80x9d.
In addition to its utility for early diagnosis of mycobacterial disease in a subject prior to the development of radiographic or bacteriological evidence of the disease, the present invention also provides for the first time a surrogate marker that can be used in an inexpensive screening method in individuals at heightened risk for developing TB. This utility was discovered by applying the approach described herein to analyze antibody responses of HIV-infected TB patients (HIV/TB) to the secreted antigens of Mt during different stages of disease progression. A majority of the HIV/TB patients had detectable antibodies to the secreted antigens of Mt for months, even years, prior to the clinical manifestation of active tuberculous disease. These patients are termed xe2x80x9cHIV/pre-TBxe2x80x9d. However, compared to the TB patients not infected with HIV (designated xe2x80x9cnon-HIV/TBxe2x80x9d), HIV/TB patients had significantly lower levels of antibodies which showed specificity for a restricted repertoire of Mt antigens. Antibodies to the 88 kDa antigen mentioned above were present in about 75% of the HIV/pre-TB sera patients who eventually developed clinical TB. HIV/TB patients who failed to develop anti-Mt antibodies did not differ in their lymphocyte profiles from those that were antibody-positive. These discoveries led to the invention of a serological surrogate marker for active pre-clinical TB in HIV-infected subjects as well as in any other high risk population. Thus, this invention provides for the first time a method for early detection of Mt infection in immunocompromised subjects. Exploitation of this discovery should make a significant contribution to the early detection of the tubercular disease and will permit a more rapid institution of therapy.
The present invention is directed to a method for the early detection of a mycobacterial disease or infection in a subject, comprising:
(a) before the onset of symptoms identifiable as clinical disease, obtaining a biological fluid sample from the subject; and
(b) assaying the sample for the presence of antibodies specific for one or more early Mt antigens, wherein detection of the antibodies is indicative of the presence of the disease or infection.
The early antigen used above may be in the form of a fraction of the lipoarabinomannan-free culture supernatant of Mt having one of the following groups of characteristics:
(a) a fraction, having a molecular weight range of about 18-45 kDa and including an approximately 42 kDa glycoprotein reactive with anti-MPT32 antibody; or
(b) a fraction having a molecular weight range of about 18-94 kDa and including an 88 kDa protein that is not reactive with mAb IT-42 or IT-57 and is characterized as having the amino acid sequence SEQ ID NO:106.
A preferred embodiment of the above method includes, prior to step (b), the step of removing from the sample antibodies specific for cross-reactive epitopes or antigens between the proteins of Mt and the proteins other bacterial genera, such as by immunoadsorption of the sample with E. coli antigens.
In the above, methods, one or more of the early antigens is preferably a secreted Mt protein or glycoprotein selected from the group consisting of
(a) an 88 kDa protein having a pI of about 5.2 characterized as having the amino acid sequence SEQ ID NO:106, which may be, but is not necessarily, obtained as a secreted protein present in Mt lipoarabinomannan-free culture filtrate;
(b) a protein characterized as Mt antigen 85C;
(c) a protein characterized as Mt antigen MPT51;
(d) a glycoprotein characterized as Mt antigen MPT32;
(e) a 49 kDa protein having a pI of about 5.1 corresponding to a spot identified as Ref. No. 82 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11; and
(f) a mixture of any one or more of (a)-(e).
In one embodiment, the method for the early detection of the presence of a mycobacterial disease or infection in a subject, comprises:
(a) before the onset of symptoms identifiable as clinical disease, obtaining a biological fluid sample, preferably serum, urine or saliva, from the subject
(b) assaying the sample to detect the presence of antibodies specific for one or more early Mt antigens selected from the group consisting of
(i) a Mt 88 kDa secreted protein having a pI of about 5.2, characterized as having the amino acid sequence SEQ ID NO:106;
(ii) a protein characterized as Mt antigen 85C;
(iii) a protein characterized as Mt antigen MPT51; and
(iv) a 49 kDa protein having a pI of about 5.1 corresponding to the spot identified as No. 82 in one or more of FIGS. 15A-F, FIG. 18, Table 9 or Table 11.
In the foregoing methods, the subject is preferably a human. In one embodiment, the subject is preferably a human infected with HIV-1 or at high risk for tuberculosis. A high risk subject includes an HIV infected individuals, an intravenous drug user, a prison inmate, a homeless person, a resident of a slum area or a household contact of a TB patient.
The present invention also includes a method for the early detection of mycobacterial disease or infection in a subject, comprising:
(a) before the onset of symptoms identifiable as clinical disease, obtaining a biological fluid sample from the subject;
(b) optionally, culturing the biological fluid under conditions permitting the growth of mycobacteria and obtaining a culture supernatant or fraction thereof;
(c) assaying the sample of step (a) or the culture supernatant or fraction of step (b) for the presence of an early Mt antigen using an antiserum or mAb specific for the early antigen;
wherein detection of the antigen is indicative of the presence of the disease or infection.
Also provided is a method for the early detection of the presence of a mycobacterial disease or infection in a subject, comprising:
(a) before the onset of symptoms identifiable as clinical disease, obtaining a biological fluid sample from the subject;
(b) assaying the sample for the presence of immune complexes consisting of one or more early Mt antigens complexed with an antibody specific for the antigen,
wherein detection of the immune complexes is indicative of the presence of the disease.
The present invention is further directed to an antigenic composition useful for early detection of Mt infection or disease comprising a mixture of two or more early Mt antigens substantially free of other proteins with which the early Mt antigens are natively admixed in a culture of Mt and which other proteins are not early Mt antigens. In a preferred embodiment, of the composition the two or more early antigens are selected from the group consisting of
(a) an 88 kDa protein having a pI of about 5.2, characterized as having the amino acid sequence SEQ ID NO:106;
(b) a protein characterized as Mt antigen 85C;
(c) a protein characterized as Mt antigen MPT51;
(d) a glycoprotein characterized as Mt antigen MPT32; and
(e) a 49 kDa protein having a pI of about 5.1 corresponding to a spot identified as Ref. No. 82 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11.
The foregoing composition may be further supplemented with one or more of the following Mt antigenic proteins:
(i) a 28 kDa antigen corresponding to the spot identified as Ref. No. 77 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11;
(ii) a 29/30 kDa antigen corresponding to the spot identified as Ref. No. 69 or 59 in FIG. 15A-F, FIG. 18, Table 9 or Table 11;
(iii) a 31 kDa antigen corresponding to the spot identified as Ref. No. 103 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11;
(iv) a 35 kDa antigen corresponding to the spot identified as Ref. No. 66 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11 and reactive with mAb IT-23;
(v) a 42 kDa antigen corresponding to the spot identified as Ref. No. 68 or 80 in FIG. 15A-F, FIG. 18, Table 9 or Table 11;
(vi) a 48 kDa antigen corresponding to the spot identified as Ref. No. 24 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11; and
(vii) a 104 kDa antigen corresponding to the spot identified as Ref. No. 111 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11.
In the present composition, any one of the early Mt antigens may be a recombinant protein or glycoprotein, preferably produced in a mycobacterial or eukaryotic expression system.
The antigenic protein may comprise either
(a) an 88 kDa protein having a pI of about 5.2 characterized as having the amino acid sequence SEQ ID NO:106;
(b) a 49 kDa protein having a pI of about 5.1 corresponding to the spot identified as Ref. No. 82 in FIGS. 15A-F, FIG. 18, Table 9 or Table 11; or
(c) a mixture of (a) and (b), wherein the protein, glycoprotein or mixture is substantially free of (i) other early Mt antigens and (ii) other proteins or glycoproteins with which it is natively admixed in a culture of Mt.
The present invention also provides a kit useful for early detection of Mt disease or infection comprising an antigenic composition as described above in combination with reagents necessary for detection of antibodies which bind to the early Mt antigen or antigens. Preferably, in the above kit, the early Mt antigen is a recombinant protein or glycoprotein. The kit may further comprise one or more of the following Mt antigenic proteins listed above as (i)-(vii).
Also provided is a kit useful for early detection of an antibody specific for an early Mt antigen in a subject, comprising (a) an early Mt antigen as described herein; and (b) one or more reagents necessary for detection of antibodies which bind to the early Mt antigen or antigens. The kit may also include at least one mAb specific for an epitope the early antigen.
In another embodiment, this invention is directed to a method for obtaining a desired mAb useful (i) for detecting an early Mt antigen or anti-Mt antibody in a sample or (ii) for isolating an early Mt antigen or epitope, which method comprises:
(a) isolating a Mt early antigen by biochemical purification or by recombinant expression;
(b) using the early antigen of step (a) to generate a collection of monoclonal antibodies each of which is specific for an epitope of the early antigen;
(c) screening the collection of monoclonal antibodies for the desired mAb which competes with
(i) a patient antiserum or antibody preparation containing an early antibody
(ii) a preexisting mAb specific for the early antigen for binding to the mycobacterial early antigen and selecting hybridoma cells which produce the competing mAb;
(d) growing the selected hybridoma cells and collecting the desired mAb produced by the cells; thereby obtaining the desired mAb.
The foregoing method is used for obtaining a mAb that is employed in an immunoassay that detects an early mycobacterial antigen or an epitope thereof, which assay comprises incubating the above mAb with a biological fluid sample suspected of containing the protein or epitope and measuring the binding of the mAb to the protein or epitope in the sample. In another embodiment, the immunoassay comprises incubating the mAb obtained as above with a biological fluid sample suspected of containing the early antibody and with a mycobacterial preparation containing an early antigen for which the early antibody is specific, and measuring the ability of the sample to compete with the mAb for binding to the early antigen.